JPH05501543A - Antibody targeting of diagnostic or therapeutic agents - Google Patents

Abstract

The targeting capability of an antibody is enhanced using an antibody-enzyme conjugate and a separate soluble substrate-agent conjugate, wherein the targeted enzyme catalyzes the conversion of a soluble substrate, bearing at least one therapeutic or diagnostic agent, to a product comprising the agent, which accumulates at the target site for effective treatment or diagnosis. This method is useful for targeting any type of agent to a site to which an antibody can selectively bind, including use for imaging, e.g. tumors, infectious lesions, fibrin clots, myocardial infarctions, non-cancerous cells, damaged normal cells, atherosclerotic plaque, lymphocyte autoreactive clones, and for therapy, e.g. with drugs, toxins, immune modulators, radioisotopes or antibiotics.

Description

[Detailed description of the invention] Background of the invention of antibody targeting of diagnostic or therapeutic agents The present invention uses an antibody-enzyme conjugate and a separate soluble drug-drug conjugate to Concerning how to increase the body's targeting ability. Here, the target enzyme is at least converting a soluble matrix carrying one therapeutic or diagnostic agent into a product containing that agent. catalyze the conversion of Therefore, this drug accumulates at the target location and is not an effective treatment. diagnosis can be made. The above method involves binding to a site to which the antibody can selectively bind. , is effective in targeting all kinds of drugs. in its use tumors, infectious foci, fibrin clots, myocardial infarction, non-tumor cells, and injured normal cells. Imaging and treatment of permanent cells, atherosclerotic plaques, lymphocyte autoreactive clones, etc. included.

Coupling an antibody or antibody fragment to a radioisotope, drug or toxin , thereby targeting diagnostic or therapeutic substances to the tumor or lesion site. It is well known that The main obstacles to using such methods are , the difficulty in loading antibodies with sufficient amounts of therapeutic or diagnostic agents. It was. A further complication is that overloading antibodies with therapeutic or diagnostic agents can lead to This leads to the body's rejection reaction and the destruction of its antibody complexes.

It is well known how cytotoxic drugs can be conjugated to antibodies to achieve the desired therapeutic effect. It is. For example, when methotrexate (MTX) is conjugated to an antibody, some It is known that selective cytotoxicity is observed. Cytotoxicity of such complexes It is desirable to enhance this by increasing the loading of the cytotoxic agent.

However, binding a large number of individual drug molecules to a single antibody ultimately leads to This will reduce the immune activity, but on the other hand, the effect will be lower than that of about 10 or more drugs. observed when drug molecules are loaded.

Another method is to bind the drug to a polymer carrier, and then bind this to an antibody. Suggestions have also been made. This means that a larger number of drug molecules can be delivered to the target site. It has the advantage of The use of polylysine as a polymeric carrier has been reported by RIrer et al. , Proc.

Natl, Acad, Sci, USA, 75:3867-3870. 1978 reported by. According to the authors' findings, approximately 13 MTXL could not be loaded, and immunoreactivity was low.

Furthermore, the high amine content of this polymer (mostly in the form of charged ammonium groups) ), the complex attaches to normal cells and has cytotoxic effects. impairs selectivity for use.

Rovland US Patent No. 4. No. N6.722 opens the following antibody conjugates: In this conjugate, multiple cytotoxic drug molecules have a molecular weight of 5.0. IIL5011. IIQ (covalently bonded to the polymeric carrier of l, and this loading The carrier is formed by randomly attaching to pendant amines or carboxyl groups. It is covalently bound to the antibody. Gho++ et al.], N1t1. Cxn ee+ Ink, 61:657-676゜1978. Other antibody-conjugated cytotoxic agents are disclosed. 5hih et al., United States Patent No. 4.699.7114 discloses methotrexate-loaded aminodext The site-specific attachment of Ran to antibodies is disclosed.

Targeted neutron activated radiotherapy is described, for example, by Goldeobe B et al.

Proc, Natl Acad, Sci, USA, III:560 (1911 4); Hxvlbornt et al.

J, Med, Chum, 15:449 (1972); Goldenbs+ g, U.S. Patent Nos. 4.332.647, 4.348.376, 4. 361°544.

No. 4.468.457, No. 4.444.744, No. 4.460.459 and and No. 4.46t1.561, as well as related family applications, U.S. Pat. Application No. 609.607 (5-14-84 application) and No. 633.999 (7- No. 24-84), the entire disclosure of which is incorporated herein by reference in the main specification. It will be included in the book.

The above reference specifically describes the boron-10-containing addition factor (aOdend+), e.g. For example, carboranes (e.g., bound to phenyldiazonium ions) and Discloses methods of incorporating into antibody conjugates using binding to the body. This zygote is , which is suitable for incorporating a relatively small number of boron-10 atoms.

Typically, 10 to 120 B-10 atoms are present, depending on immunoreactivity and the amount of recovered product. The carborane-phenyldiazonium junction Attach to IgG using a method. For effective treatment, large numbers of B-10 atoms are required. It is desirable to be able to target the tumor site.

Therefore, by overloading the targeting antibody with the drug, the immune response is a sufficient amount of treatment without loss of responsiveness and/or while inducing an immunogenic response. Antibody targeting methods that allow therapeutic or diagnostic agents to be attached to target sites There continues to be a need to do so.

Purpose of invention One objective of the present invention is to target antibodies by amplifying targeting events. The goal is to provide a way to emphasize targeting capabilities.

Another object of the present invention is to treat cancer, infectious lesions, or the like, such as myocardial infarction. It is an object of the present invention to provide a drug effective for the diagnosis and/or treatment of other lesions.

Yet another object of the invention is to provide therapeutic or diagnostic agents without the need for highly loaded antibodies. The goal is to allow drug withdrawal to accumulate to a high degree at the target site.

Yet another object of the invention is to provide thermally neutral Acts as an effective therapeutic agent against tumors and lesions for child-activated radiotherapy The objective of the present invention is to provide a method for targeting a large enough number of boron atoms.

Other objects of the invention will be apparent to those skilled in the art in light of the following discussion. cormorant.

Summary of the invention The above and other objects of the present invention are to deliver diagnostic and/or therapeutic agents to target sites. This is achieved by providing a way to target (a) Targeting the antibody-enzyme conjugate and enzymatic activity. parenterally injecting into the mammal an amount effective for However, this antibody 0 that is reactive with at least one antigen present in (b) the antibody-enzyme conjugate localizes to the target site and is substantially cleared from the mammalian circulatory system; After sufficient time has elapsed for the soluble substrate-drug conjugate to be released into the target area. parenterally injecting into the mammal an amount effective to cause the mammal to adhere to the site. However, this The conjugate is converted by the enzyme to form a product containing the drug, and this product accumulates at target sites for effective treatment and/or diagnosis. Also, the substrate - the drug conjugate comprises a substrate for said enzyme and is associated with at least one diagnostic or therapeutic agent; combined.

In addition, the above-mentioned enzyme or the substrate/drug complex has a similar activity. Either at a non-target site along the route of administration or at the substrate/drug. Interfering with the targeting and accumulation of the drug in the biodistribution of the drug complex It is not endogenous to the mammal in any significant amount.

The present invention also provides reagents, sterile injectable formulations, and and a kit.

detailed description Conventional techniques involve applying therapeutic or diagnostic agents directly to antibodies or to carriers conjugated to antibodies. Discloses a method for combining. Problems associated with binding drugs to antibodies cross-linking, loss of immunoreactivity, immunogenicity, insufficient loading of the drug onto antibodies. , and inappropriate attachment of the drug to the target site. The present invention provides antibody-enzyme conjugation The use of a separate substrate-drug conjugate to solve these problems This allows the antibody to be loaded with a diagnostic or therapeutic agent onto the antibody. It can be targeted to the desired area without the need for

The method of targeting the diagnostic or therapeutic agent of the present invention to a target site first involves The antibody-enzyme conjugate is administered parenterally to a mammal in an amount effective for targeting and enzymatic activity. injection and that the conjugate is localized at the target site and in the circulation of the mammal. This is achieved by waiting for sufficient time to have passed for the substance to be substantially removed from the system. will be accomplished. The next step in the method of the invention is to obtain an effective amount of soluble material to attach to the target site. The substrate-drug conjugate is injected parenterally into the mammal. This complex can be converted by its enzymes into a product containing the drug, and this product Accumulate at the site to achieve effective treatment or diagnosis. This substrate-drug conjugate A substrate of the enzyme coupled to at least one diagnostic or therapeutic agent.

The antibody component of the antibody-enzyme conjugate is the targeting moiety that selectively binds to at least one antigen present in the target site . The enzymatic component of this complex is therefore localized to the target site. Once the non-target Once the conjugate has been substantially removed from the bloodstream, inject the substrate-drug conjugate. . This substrate-drug complex may contain more than a negligible amount of antibody-enzyme complex or or endogenous enzymes with similar effects on the way to the target site. stomach.

However, once this substrate-drug conjugate reaches the target site, the complex Enzymes convert it into products that include diagnostic or therapeutic agents. This enzyme has a substrate - convert multiple molecules or subunits of the enzyme conjugate and deliver multiple products to the target site It is released in a form that is easy to accumulate. This involves the target site and the body fluids surrounding the tissue or This is because partitioning with other antigen-containing media present at the target site itself favors accumulation. be. This enzyme thus amplifies the targeting ability of antibodies, but also Does not require conjugation to targeting antibodies. The drug also targets the target site. , where a diagnostic or therapeutic action can be carried out.

book Unless otherwise specified, use of the term “antibody” refers to antibody fragments. and therefore equivalent to the term “antibody/fragment”. Therefore, in this explanation, the two are used interchangeably. Antibodies can be of any class For example, whole immunoglobulin of IgG, Igll, IgA, IgD, IgE hybrids with dual or multiple antigen or epitope specificity. Fragments containing hybrid fragments, such as F (gb’), F(ab)2. It may also be Fib', Fab, etc.

Antibodies include antiserum preparations, preferably affinity purified and highly immunological. have a binding constant of at least about 10 to 17 moles, preferably Preferably, those with immunoreactivity of at least about 1091/mol+, those with high immunological properties. having isomerism, e.g. at least about 40%, preferably less (at least about 60%) , more preferably with an immunospecificity of about 75-95%, with other tissue antigens. Those with low cross-reactivity, for example, about 30% or less, preferably about 15% or less, and The cross-reactivity is preferably about 5% or less. This antiserum is usually Affinity purification can be performed by the following method. For example, an antigen For example, the column is coupled to a chromatography column packed with Sephadex. The antiserum is passed through the membrane, thereby retaining specific antibodies and other immunoglobulins. The purified anti-oxidant is then purified by elution with a chaotropic agent. Collect the body and further purify it if necessary.

Monoclonal antibodies are also suitable for use in the method of the invention and their high Preferred for specificity. Monoclonal antibodies are now a routine procedure i.e., immunization of mammals with immunogenic antigen preparations, immunolymphatic or pancreatic cells. Fusion of cells with immortalized myeloma cell lines and isolation of specific hybridoma clones It is easily prepared by More special preparation methods for monoclonal antibodies, examples For example, interspecies fusion (1nte mouth peeiCi1osion+), highly variable region, Genetic engineering operations cannot be ruled out. This reason may affect the effectiveness of the method of the present invention. This is because the main reason for this is the antigen specificity of the antibody.

Antibody fragments are described, for example, in US Pat. No. 4,331,647, among others. Whole immunoglobulins are treated with pepsin or protein by conventional methods disclosed. Can be made by digesting with pine.

Target sites include cancer, infections and parasitic lesions, fibrin clots, myocardial infarction, and atherosclerotic plaques. Lark, injured normal cells, non-cancerous cells, and lymphocyte autoreactive clones may be used, but is not limited to these.

caused by tumors or foci of infection, including viral, bacterial, fungal, and parasitic infections. Many antibodies and antibodies that specifically bind to markers produced or associated with and antibody fragments, as well as antigens and products associated with such microorganisms. In particular,) Ian+en et al., U.S. Patent No. 3.927.193; oldenb+rg.

U.S. Patent Nos. 4.331.647 and 4.348.376.

No. 4.361.544, No. 4.468.457, No. 4.444.744 issue.

Nos. 4.460.459 and 4.460.561 and related pending proceedings. No. 609.607 and US Pat. No. 633.999. It is. All such disclosures are hereby incorporated by reference in their entirety. I will do so.

Anti-fibrin antibodies are well known in the art.

Antibodies targeting myocardial infarction are, for example, I(abet), US Pat. No. 4.0. No. 35.945, the disclosure of which is incorporated herein by reference in its entirety. I will include it in the book. Antibodies that target normal tissues or organs can be used, for example, in It is disclosed in U.S. Patent No. 4.735.210, and the disclosure content is referred to It is incorporated herein as is. Anti-fibrin antibodies are important in this field. So, what is the best antibody to bind to atherosclerotic plaques and lymphocyte autoreactive clones? well known.

In general, antibodies are typically raised against any antigen using a number of conventional techniques well known in the art. It is also possible to cause this to occur.

found in sufficient concentrations in a site in the mammalian body of diagnostic or therapeutic interest. Any method can be used to target antibodies against the antigen that is released. can be used to produce the antibody-enzyme conjugate used in the method of the invention.

Furthermore, at least one enzyme that is specific for the antigen present in the target site one binding site and at least another specific for the V element component of the antibody-former conjugate. Bispecific antibodies/fragments having more than one binding site can be used in the method of the invention. It should also be noted that it is possible to Such antibodies should be fermented before injection. This eliminates the need to covalently attach the enzyme to the antibody. can be avoided. Alternatively, the antibody can be injected and substantially removed from the circulatory system. Enzyme is present in sufficient quantities to reach and bind to the localized antibody. Amounts and routes by which antibody-enzyme conjugates can be formed in situ (in day + u) The enzyme can be injected at

Bispecific antibodies can be made by a variety of conventional methods.

For example, by disulfide cleavage, a mixture of total 1 gG or preferably F( xb') fusing one or more clones by reconstitution of a mixture of two fragments to form polyomas that produce one or more specific immunoglobulins. Especially? and can be prepared by genetic engineering. dual singularity Antibodies may bind to one or more epitopes on an enzyme, but do not interfere with enzyme activity. It must not bind to the site where it is present.

The enzymes used in the invention convert substantially soluble substrate-drug conjugates into diagnostic drug and/or therapeutic agent and which accumulates at the target site. Must be. Both of the above enzymes and enzymes with similar substrate specificity are The route of administration or biodistribution of the quality-drug conjugate is such that it is endogenous to the mammal. Must not be. Otherwise, the drug will be released at a site other than the target site. , this is usually, but not always, the diagnostic or therapeutic action of the drug. It would hinder efficiency and make it unstable.

In principle, an enzyme is one that can bind to an antibody and convert the substrate-drug conjugate into a product. Any type of enzyme may be used, but the above precautions apply to this as well. be done. Proteases, glycosidases, esterases, etc. are These are all general types of enzymes that can be used in the present invention. Further of suitable enzymes Specific examples include glucuronidase, beta-glucosidase, Cutamase, cellulase, dextranase, fructase, aminopeptidase These include, but are not limited to, zeolin, lysozyme, and the like.

The enzyme is selected as a function of the type of substrate-drug conjugate chosen. For example, the substrate Choosing dextran as an enzyme means using dexranase as an enzyme. It is connected with. Similarly, cellulases are used with cellulosic substrates. Substrate- Using glucuronide as a drug conjugate requires glucuronidase as an enzyme. It is associated with use, etc.

Apart from forming antibody-enzyme conjugates in situ, the enzyme can be covalently attached to the antibody. It is advantageous to do so. i.e. directly combined or short or via a long linker moiety or one or more linkages on the antibody and/or enzyme. Functional groups such as amino groups, carboxyl groups, phenyl groups, thiol groups, hydroxyl groups, etc. Bonding via a syl group is advantageous. Various commonly used linkers - 0 examples For example, dathiocyanates, diisothiocyanates, bis(hydroxysuccinates) (imide) esters, carbodiimides, maleimide-hydroxysuccinimide Midesters, glutaradehyde, etc. can be used.

A simple method is to mix the antibody with the enzyme in the presence of glutaraldehyde, and the antibody-enzyme It is to form a zygote. The first S+hi[+ base bond is e.g. It is possible to stabilize it by Eastoniumin conversion by dryide reduction. child The method is typically performed using peroxygens used for example in immunohistochemistry or immunoassays. Used in the preparation of Dase-antibody conjugates. diisothiocyanate or carbodi Imides may be used in place of glutaraldehyde.

More selective linkages can be achieved with heterobifunctional linkers such as maleimide-hydroxy This can be achieved using succinimide esters. When this substance is reacted with an enzyme, Inducing an amino group on the enzyme. This derivative may further include, for example, free sulfhydryl Antibody Fab fragments with drill groups (or even larger fragments) or intact immunoglobulins, which can be cured by e.g. hydryl groups).

It is advantageous to bind the enzyme to the antibody at a site far removed from the antigen binding site. Ru. This applies, for example, to the cleaved sulfhydryl group, as mentioned above. This is achieved by binding to Another method is to remove the carbohydrate portion in advance. By reacting the oxidized antibody with an enzyme having at least one free amino group, be. With this, the first 5chif! A base (imine) bond is obtained. this The bond is stabilized by reduction to a secondary amine, for example by borohydride reduction, and Preferably, a terminal zygote is formed.

Because of conjugate size issues, one antibody is usually coupled to one enzyme molecule. It is preferable to However, multiple antibody fragments, e.g. F+b or combines the F(ib’)2 fragment to a single enzyme to target the antigen. It would also be advantageous to increase the binding affinity or efficiency of binding. Alright If the enzyme is not too bulky, multiple enzyme molecules can be combined with a single antibody. or to antibody fragments to increase conjugate turnover and reach the target site. It would be useful to increase the rate of deposition of diagnostic or therapeutic agents. 1 or more types The conjugate of the enzyme and the antibody also allows it to reach the target site and less Can be used unless promptly removed. a mixture of zygotes of various sizes, or Conjugates containing aggregates can also be used as long as the precautions just mentioned are followed.

Additionally, the antibody-enzyme conjugate can be labeled with radioisotopes or magnetic resonance image enhancers. or combine them with, or modify them so that they can be joined. You can also do it. This monitors the clearance of the conjugate from the mammalian circulatory system. or to ensure that the substrate-drug conjugate is sufficiently localized at the target site prior to administration. You can check whether Alternatively, the conjugate may be labeled, e.g. radioactive. A sex label, a fluorescent label, etc. can also be added. This allows blood and urine to be removed. It is now possible to detect and quantify the conjugate in body fluids, and therefore to measure and/or estimate targeting and/or clearance. becomes possible.

Conventional radiolabeling methods suitable for labeling proteins for in vitro use Both are generally suitable for labeling antibody-enzyme conjugates and are also suitable for labeling substrates. - It is also often convenient to label enzyme conjugates. About this below This can be achieved, for example, by direct labeling with l-131, l-123, as described , by metallization, e.g. with Tc-99m or Cu ions, by conventional techniques. Therefore, or to combine chelating agents for radioactive metals or paramagnetic ions. This can be achieved by

Such chelating agents and methods of conjugating them to antibodies are well within the skill of those skilled in the art. known and, inter alia, for example the Goldenbe+g patent and C. hilds et al.], Hue, Med, 26:293 (1985). There is.

The substrate-drug conjugate includes a substrate that binds to the enzyme localized in the antibody-enzyme conjugate. Therefore, it can be converted into a product. A drug is a diagnostic or therapeutic agent that targets a specific area. Drug targeting to the drug would be advantageous for its efficacy. like that Therapeutic and diagnostic agents include, for example, toxins, antibiotics or chemotherapeutic agents, radioisotopes. elements, paramagnetic ions, boron addition factors, cytokines, photosensitizers, radiosensitizers, These include vasodilators.

The substrate-drug conjugate must be soluble for administration and delivery to the target site. No. Moreover, this one reaches the target and is attracted to the site rather than the zygote. must be converted to a product with a substantially more favorable partition coefficient. Book As used herein, the term "soluble" means that the conjugate is administered into a fluid and that A diagnostically or therapeutically effective amount of the fluid is carried by the fluid to the target site. This means that the conjugate can be sufficiently lysed to be delivered to the target site. one In general, administration is done quietly. The conjugate is administered intravenously or intraarterially into the bloodstream, and the conjugate is dissolved in the serum and preferably or is sufficiently hydrophilic that it is mostly carried by the aqueous phase of serum; Diffuse readily through the vessel wall and into the intercellular fluid, if this is necessary. desirable in some cases.

Of course, cardiac imaging or arteriosclerotic plaque imaging where the target site is in the circulatory system. In the case of imaging or treatment, water solubility/lipid solubility means that the Motoga-drug conjugate is Thus, with cleavage, target reduction is of no significance. Targeting machine of the present invention It is precisely this distribution from the drug that characterizes the The drug-drug conjugate is acted upon by the enzyme component of the targeting antibody-enzyme conjugate. then the drug accumulates significantly more than the substrate-enzyme conjugate would accumulate in the absence of the enzyme. Accumulates in large quantities at target sites. This is how the drug is separated.

This includes some common examples and more detailed information about the various types. It will be easier to understand if you look at the explanation.

A general method for making the substrate-drug conjugates of the invention includes at least one therapeutic agent or involves covalently linking a diagnostic agent to a substrate.

Certain cytotoxic agents useful in anti-tumor therapy are relatively insoluble in serum. Non I got addicted to zygote things (some of them are poisonous, but if you convert them to zygote, they become toxic) It decreases considerably. Relatively poorly soluble drugs can be combined with more soluble conjugates, such as glucuronides. Conversion to venous, arterial, and capillary fluid increases its solubility in the aqueous phase of serum. Increases permeability through the cell walls of blood vessels and improves access to the intercellular fluid surrounding serious injuries. Ru. In fact, certain toxic substances, such as aromatic or cycloaliphatic alcohols, Substances such as alcohols, phenols, and amines are converted into glucuronides in the liver. Conversion is the body's detoxification method for these substances, thereby also It is also easier to excrete in the urine.

The drug is attached to glucuronic acid to form a glucuronide, which dissolves the conjugate. Ru. The bond is usually to a hydroxyl, thiol or amine group on the drug. carried out, the aldehyde carbon of this glucuronic acid, acetal, thioacetal , forming an aminoacetal. This conjugate produces an antibody-enzyme at the target site. It is cleaved by the enzyme glucuronidase, which is an enzymatic component of the conjugate. Then this Free drugs become almost insoluble in the intercellular fluid and tend to adhere to the cell membranes of surrounding cells. and exerts cytotoxic effects at the localized site of the antibody-enzyme conjugate.

One method for preparing such glucuronides is to use mammals such as cows, goats, The drug is injected into a horse or primate. Some drugs may cause It is converted into glucuronides in the human liver, and this drug-glucuronide conjugate is found in the urine. Excreted. This drug is administered by a slow hepatic pump from the hepatic artery or portal vein. Preferably, it is administered by intravenous infusion. Next, urine collection and glucuronide conjugate Extraction can be carried out, for example, by ion exchange chromatography. Also, another As a method, UDP-glucuronic acid is reacted with the drug and then the glucuronide is reacted with the drug. There is a way to isolate it from the reaction mixture. This reaction was isolated from the endoplasmic reticulum of mammalian liver. and/or the reaction can be carried out under the catalysis of an enzyme that It can be carried out in the presence of extracts or homogenates.

One class of antitumor agents that can be converted to such substrates is evirubicin, or doxo- The 4-epimer of rubicin is an anthracycyl glycoside. and human β-D-glucuronidase (^+csmooe, C1nce+ Res, 45:5995. (1985).

Other analogs with fewer polar groups are expected to have even higher fat solubility. , and much greater performance is expected with such a method. Other drugs and toxins , boron compounds, or aromatic or alicyclic alcohols, thiols, amino Chelating agents with groups are also candidates for such conjugate formation.

Another type of substrate-drug conjugate involves linking multiple drugs intermittently along the polymer backbone. It is a combined polymer. This polymer is highly sensitive to the enzyme component of the antibody-enzyme complex. or a segment that is a substrate for such an enzyme. It may also include branch schools. The drug molecule is attached to the polymer as follows: be done. That is, the drug is released from the polymer unit by enzymatic cleavage, or have the necessary low solubility or target site cells, tissues, or diseases. Compared to the fluid surrounding a location such as a nest component, the distribution coefficient to such a location is higher. so that the drug bound to a sufficiently small number of units is cleaved to be preferred. is combined with

Examples of polymers used in this way include, for example, polyols, polysaccharides, Examples include polypeptides. An example of a polysaccharide is dextran, i.e. Rufar glycoside, which is cleaved by the enzyme dextranase. diagnosis The agent or therapeutic agent is a reactive group sensitive to the hydroxyl groups of dextran, e.g. Functionalized to include baric anhydride layer, isocyanates, isothiocyanates, etc. can be converted into Alternatively, dextran can be added in several ways, e.g. It can also be derivatized by converting to aminodextran.

A method for producing a substrate-drug conjugate containing an aminodextran (AD) carrier usually involves the following steps: Dextran polymer advantageously has an average molecular weight of about 10.000-LOG,000 amount (MW), preferably about 10.000-40.000, more preferably Dextran with an average molecular weight of about 15,000 is the starting material. Then this Dextran is reacted with an oxidizing agent, and some of its carbohydrate rings are oxidized to aldehyde. Forms a hydride group. This oxidation is carried out in a conventional manner using a glycolytic reagent such as Ni104. It is convenient to do this by.

The amount of oxidizing agent is approximately 50-1 per dextran having a MW of approximately 40,000. At the same time also other The preparation was made so that approximately the same proportion of aldehyde groups were generated for MW dextran. It is convenient to arrange it. The reason for the increase in the number of aldehyde groups that later become amine groups is , the polymer then behaves like polylysine and at the same time resists enzymatic cleavage. This is disadvantageous because it exhibits resistance. When the number is lower than this, drugs, toxins, The loading amount of the chelating agent or boron addition factor will be lower than the desired value, This is particularly disadvantageous when the turnover rate of the enzyme is low.

The oxidized dextran is then added to a polyamine, preferably a diamine, and more preferably a diamine. or with mono- or poly-hydroxydiamines. with a suitable amine For example, ethylene diamine, propylene diamine or similar polymethyl Diamines, diethylenetriamine or similar polyamines, 1.3- Diamino-2-hydroxypropane or other similar hydroxylated diamino-2-hydroxypropane Examples include amines and polyamines. Excess relative to aldehyde group amine to ensure that almost all of the aldehyde groups are removed by 5chif! base (imine) group.

Reductive stabilization of the resulting intermediate can be achieved by treating this Sch101 base intermediate with a reducing agent, e.g. B) It can be carried out by reacting with 1, NaBI (3CM, etc.). The base agent ensures that almost all of this imine group is reduced to secondary amines. If there are any unreacted aldehyde groups, convert them to hydroxyl groups. Definitely give back. The resulting adduct is further passed through the column with the normal sieve fraction passing through the column. It can be purified by removing cross-linked dextran. The first occurrence on AD For the number of monoamino groups, react the weighed sample with trinitrobenzene sulfonic acid. can be estimated by correcting the optical density at 420 nm using a standard material. Ru. This method usually almost completely reduces the calculated number of aldehyde groups to the first Converted to amine group.

Alternatively, dextran can be derivatized using conventional methods to introduce amine groups. Wear. For example, it may be reacted with cyanogen bromide and then with a diamine.

AD can be caused by certain drugs, toxins, chelating agents, or derivatives of boron-added factors. and must be reacted with its activated form, preferably a carboxyl-activated derivative. No. This active form can be obtained by conventional means such as dicyclohexylcarbodiimide ( D CC) or a water-soluble variant thereof.

Methotrexate (MTX) is the standard used in producing the conjugate of the present invention. Although a typical drug, it is used to illustrate one of the procedures of the invention. So Similar procedures are appropriate for other drugs, toxins, chelators, and boron-adding agents. However, such changes may be obvious to those skilled in the art. cormorant. Activation of MTX is performed with any conventional carboxyl activating reagent such as DCC. It's convenient. I can do it. If necessary, N-hydroxysuccinimide ( HO3u) to form an activated ester. This reaction is usually polar, non-polar Protic solvents such as dimethylformamide (DMF), dimethyl sulfur It is carried out in oxide (DMSO), etc. Other activated esters, such as P-ni Trobenzoate and the like can also be used, as can mixed acid anhydrides. D.C.C. /HO3U activation is mild and activated MTX can be reacted with AD in aqueous solution. This is preferable because it allows

The ratio of activated MTX to AD is such that about half of the amino groups present on AD are reacted. and forms an amide bond with the carboxyl group of activated MTX. It is preferable. Thus, about 100 amine groups have a starting MW of about 40. 000 AD, up to about 50 of these must react with activated MTX. Must be. If a ratio of about 50:I MTI:AD is used, typically about 25- Introduce 50 MTX molecules. It is difficult to increase the load higher than this, which is This is because the adduct begins to precipitate due to its increased insolubility.

Examples of applications used in other drugs include 5-fluorouracil (5-FU). If we were to list the load depending on the oxidation using periodate and reacting this intermediate with aminodextran; Furthermore, it can be implemented by reductively stabilizing this 5chitf base adduct. Wear. Cyclohequinimide can be loaded as follows. i.e. , directly reacting the carbonyl of cyclohexane with the amine group of aminodextran. or by reductively stabilizing the side chain hydrocarbons. xyl with excess diisothiocyanate linker and the isothiocyanate By reacting cyanate derivatives with amines on aminodextran, Alternatively, the imide nitrogen can be reacted with e.g. a halo acid or halo ester, and then , the obtained carboxyl derivative is reacted with, for example, DCC, and aminodex It can be loaded by condensation with an amine on tolan.

Another example comes from the antibiotic mitomycin C and its analogs. this The molecule has an amine group and a cyclic imine, either of which can be an alkylated activating group. For example, succinimidyloxyiodoacetic acid or sulfosufuccinimidyl Oxy(4-iodoacetyl)aminobenzoate (sulfur 5IAB) and can be reacted. Next, using the obtained intermediate, aminodextran Alkylate the amine group on top. Alternatively, carboxyl groups can be added, e.g. introduced using citric acid, then activated using e.g. DCC, and this activated intermediate are combined in the same manner as above.

Toxins, such as pokeweed antiviral protein (PAP) or Syn A chain etc. are activated by glutaraldehyde condensation or by the activity on the protein. Reacting the carboxylic acid group with the amine on aminodextran to bond Can be done.

In addition to the specific types listed above, there are also tumor cells and microorganisms that can infect humans and cause disease. Many drugs and toxins are known that have cytotoxic effects on substances. the You can find things like this in drug and toxin manuals, such as the Merck Index. be able to. All such drugs are well known in the art and described above. It can be attached onto AD by the usual methods suggested by the similarity with.

According to the invention, a drug can be administered as a complex, partially or completely in its circulation. Being able to make the drug non-toxic reduces the systemic side effects of the drug and Its use can be approved even if systemic administration of the drug is unacceptable. for example , MTX and ncloheximide are too toxic when administered systemically. There are many. According to the invention, even more molecules of this drug bound to a substrate carrier administration, it not only alleviates systemic toxicity but also allows for treatment. .

Loading of the drug onto the matrix carrier is soluble (i.e., the fluid that bathes the target site and the target cell, tissue or or other structures such as atherosclerotic plaques, fibrin clots, viral particles, (partition coefficient between parasites, etc.) have a partition coefficient to the target that is sufficiently favorable to undergo bare cleavage and exert the desired therapeutic effect. It will depend on the efficiency of incorporating the drug. Generally, drugs are negative on dextran. The monosaccharide subunit to drug ratio is preferably about 3 to about 5. Delicious. Of course, it is desirable to do so, and it is not a limited amount. . Excessive loading of drug molecules can inhibit enzyme activity.

This is primarily due to steric hindrance that prevents the substrate conjugate from binding to the active site of the enzyme. Therefore it happens. If underloaded, the drug fluid obtained as a result of enzymatic cleavage Solubility may not be reduced sufficiently. This is because drugs (perhaps still 2-3 glycosidic subunits attached to it) reduce the solubility of of the polysaccharide-drug conjugate before enough sugar subunits are cleaved and released. This is because a small portion escapes from the bound enzyme and diffuses. That is, if the drug conveniently distributed from the surrounding fluid to the target site, e.g. tumor cell membrane, bacterial cell wall , atherosclerotic plaques, or fibrin clots. This is because solubility does not decrease. Toxins are often large proteins, so they cannot be used as drugs. The loading amount can be reduced compared to other agents.

Chelating agents or magnetic resonance enhancers for radioactive metals are also well known in the field. ing. Typical are ethylenediaminetetraacetic acid (EDTA) and diethyl It is a derivative of thylenetriaminepentaacetic acid (DPTA). These are usually on the side It has a functional group on the chain by which the chelating agent is attached to the carrier. This same The same group is used to attach the chelating agent to the amine group on AD. Or chelate attach carboxyl or amine groups on the agent, resulting in AD activation or Alternatively, derivatization is performed first and then conjugation is performed. All of these are known It can be done by law. For example, deferoxa, a chelating agent for G5-67. Min has a free amine group. This amine group can be activated with a suitable linker. In addition, A Binds to the amine on D. Other methods of attaching chelating agents to the amines of AD depends on the functionality of the chelating agent, but will be obvious to those skilled in the art.

A boron addenda, e.g. carborane, binds to the substrate conjugate. activated by thermal neutron irradiation when targeted to the lesion by antibodies. sexualized and converted into radioactive atoms. This atom decays by alpha radiation, Produces highly cytotoxic local effects. Highly loaded with boron addition factor Similar to magnetic resonance enhancing ions, this is extremely important to enhance its effect. be. As is well known in this field, carborane is Made using carboxyl groups on the chain. This carborane has a carboxyl group. One of the two methods of the present invention is coupled onto AD by activation and condensation with an amine on a carrier. In embodiments, the substrate-drug conjugate contains multiple boron atoms, more preferably Preferably, it is prepared from a reagent rich in boron-10 isotope, which contains about 96% boron. Boron-containing reagents enriched in element-10 are commercially available. Such a zygote is inside , this brings a sufficient number of boron atoms to the severely injured or focal site and heats it up. It will be possible to deliver therapeutic doses of alpha particles to target tissues during neutron irradiation. It is from. Moreover, this is because the administration of the antibody-enzyme conjugate localized to the target tissue A relatively low percentage of eg 1-10% is also possible. like this The percent localization is based on the antibody-targeted species (xatibod7-tgrg+led +p+cie+) is not that unusual.

The boron-loaded substrate conjugates of the present invention have a large number of boron atoms per substrate molecule. child Usually there are at least about 50 to about 10,000, preferably about 200 to about 10,000. There are approximately 2,000 pieces. Again, these are boron-1 with a natural abundance of 20% It is more cost-effective to use a conjugate with a larger number of boron atoms, including zero isotopes. It is advantageous to be rich in boron-10, preferably about 96% boron-10. desirable.

The substrate-drug conjugate may contain boron-free moieties or boron and other useful moieties. It may also include a portion containing a group. Such groups include, for example, nuclides, l-123, 1-125, or l-131, or functional groups such as agents, chelates containing metal ions, drugs, toxins, chromophores, chromogens, fluorescent markers - etc., all of which increase the therapeutic effect or improve the effectiveness of boron-added factors. to enable deposition and/or clearance monitoring or to perform ancillary therapeutic actions. It is now. Substrate-drug conjugates may incorporate functional groups, the main The purpose is to increase the fat solubility or reduce the water solubility of enzymatic cleavage products containing boron addition factors. It's about doing things.

In making such a conjugate, it is convenient to use a boron cage compound. However, this is relatively easy to handle.

Moreover, each such cage compound has 5-12 boron atoms at the target site. Depends on what you can carry. For many of the reactions discussed below, those skilled in the art will be able to , you will be able to find general references in this field. Above all, too The best and most comprehensive one is MaeNettie+ et al.'Pol! bed+tlBo+xne+', (Dekke+, New York, 196 81; Mus mouth el t’Boron Hydride Chemir trH', (Aczdemic Ptess, N + Cao Yolk.

1975); Grimes, 'Cx+bo+xnes', (Academi c Press, New York.

It is 197G+. The above literature describes the synthesis of organic derivatives containing multiple boron atoms. It contains a wealth of literature on specific topics within the broad subject matter range of . Hav lho+Ωe, U.S. Patent Application No. 742.436, (filed 6-7-85) The details are also included in this application, which is hereby incorporated by reference in its entirety. I'll do it.

Alternatives to alpha glycosides like dextran and aminodextran As a beta glycoside such as carboxymethyl cellulose (CMC) be. This is cleaved by cellulase enzymes. This CMC has diagnostic agents or therapeutic agents. The method of binding therapeutic agents is the same as for dextran; This is because both are sugar polymers and differ only in the stereochemistry of the glycosidic bonds.

Deriving additional functional molecules from CMC involves converting CMC into carbodiimide-type condensates. to form an amide bond using the amine group on the diagnostic or therapeutic agent. This would be most conveniently achieved by

Alternatively, the glycol can be gently oxidized with a cleavage reagent such as periodate to If aldehyde groups are formed at multiple sites on the mirror and then reacted with diamine, Amino CMCs can be formed that are convenient to react with various functional groups. . It is also possible to condense this oxidized CMC with an amine and stabilize it with borohydride. It is possible. Other means of attaching drugs to CMC are known to those skilled in the art. It should be obvious.

Yet another variation on polymeric substrates is that they are not cleaved by enzymes, but It has a short oligomeric linker segment that serves as a substrate for drugs and chelating agents. is a polymer with a stimulant, a boron-added agent, or a similar diagnostic or therapeutic agent. . As one example, polyvinyl alcohol and multiple short oligosaccharides For example, short dextran or cellulose oligomers (e.g. 5-50 , preferably consisting of 5-20 glycoside subunits. ) as a carrier for It can also be used. Amination of polyvinyl alcohol with e.g. cyanogen bromide Then, diamine condensation may be performed. Oligosaccharides are, for example, periodic acid Mild oxidation with salts and condensation with aminated polymers to form 5chill base bonds , which is preferably further stabilized by borohydride reduction. next, The obtained oligomer-bound polymer is combined with dextran polymer or cellulose polymer. may be lightly aminated as described above for Functionalized in a conventional manner, on each oligomer linker at least two, preferably Approximately 2-5 amine groups are attached. Next, on average 1-3 drug molecules, Agents, boron-adding agents, and other agents are attached to each of the oligomers.

It is obvious that many other variants are possible.

Aminated polymers and drugs with lightly oxidized dextran oligomer linkers Alternatively, condensation to other drugs may be performed simultaneously or sequentially. However, it will stabilize after that. Oligomerization using other functional groups on the drug or other functional groups may be used to attach the oligomer to the polymeric carrier. It may be combined with

Acrylic acid polymers may also be used, but in this case, the acrylic acid carboxyl is The amide bond formed by activation with rubodiimide Attach the xtran oligomer to the polymer. You can also use polypeptides. In this case, oligomer linkers are attached to carboxyl or amine residues on the carrier. combine. An oligosaccharide linker may also be used instead, in which case the linker Contains an esterase or peptidase enzyme that cleaves the car. Those skilled in the art are within the broad scope of the invention and can be prepared by conventional synthetic methods. Other variants may be foreseen.

Yet another approach is to use carrier polymers such as: Sunawa that contains drugs, chelating agents, boron-adding factors, or other agents, and that In the modified form, the target is solubilized and the molecule is released by the targeting enzyme. It is a polymer that is cut. Another example of such a substrate-drug conjugate is polylysine, in which multiple radioactive metals or paramagnetic metal chelating agents are added. , carborane or MTX molecules. Next, this carrier conjugate with multiple short dextran oligomers, e.g. by 5chiff base formation. condensation to form lightly oxidized dextran and borohydride stabilization ( If the chelating agent is bound to a carrier, then the radioactive isotope or loads paramagnetic ions).

This increases the solubility of polylysine (decreases its 'stickiness') and transports it in serum. The ratio is adjusted to make it easier to transport and to make it easier to pass through the capillary wall. mark Anti-tumor antibody-dextranase conjugate localized at a target site, e.g. in a certain tumor. The body takes the dextran coating from this polylysine and it becomes [sticky] again. strip away enough so that this thing adheres to the tumor cells and this binding point is removed. Lyridine has a diagnostic or therapeutic agent attached to it, so it acts on these tumor cells. enabling its imaging or cytotoxic treatment.

Highly aminated aminodextran can act as polylysine or Short oligomeric substrate linkers as discussed may also be substituted. This one is poly Like lysine, it is "sticky" to cell membranes and other tissues. Other polypeptides Polymers that are highly aminated or highly aminated can also be used as carriers to coat substrates. It can function in solubilization and solubilization. In fact, amination Not essential for body function.

This is because conjugates of certain carriers and one or more diagnostic and/or therapeutic agents are preferred. Whatever functionality results in favorable partitioning, it solubilizes the substrate oligomer. or by simply solubilizing small substrate molecules. Because you can. That is, the resulting soluble conjugate can be administered to serum or another The coated molecules circulate easily in fluids, but in the fluid that bathes the target site, the antibody-enzyme It becomes poorly soluble when it is cleaved by the action of enzymes local to the conjugate. Solubilization is performed as follows.

Loaded carrier polymer to “coating” solubilizing substrate groups or oligomers The proportions will vary depending on the nature of the target site and the characteristics of the components. If polylysine or When using something as a carrier that has a function equivalent to that, oligodextran Coating with dextran: polylyte in a ratio of about 1=10 to about 100:1 by weight gin, preferably in a ratio of about 1=1 to about 10:1, more preferably Advantageously, a ratio of about 3:1 to about 7:1 is used. One example is a MW of about 1. 500 Daltons of polylysine, each with a MW of approximately 15,000 Daltons. It is coated with about 3-7 dextran oligomers.

For example, Go! +1enb++1. U.S. Patent No. 4.624.846 As disclosed, it forms a complex with the zygote and is activated by macrophages and reticuloendothelial threads. By using a second antibody so that the rate of uptake of the conjugate is increased, After sufficient time has elapsed for the diagnostic or therapeutic agent to localize or adhere, accelerating the clearance of body-enzyme conjugates and/or substrate-enzyme conjugates; It would be convenient if it could be done. Optimal for second antibody clearance such as The time can be measured with the help of a label on either zygote, thereby , the degree of localization of the antibody-enzyme conjugate at the target site, and/or the target site The extent of drug attachment and biodistribution of non-targeted conjugates in can.

The reagents are available in dual injection formulations (dualinje+tt) for human therapy and diagnosis. It is convenient if it is supplied as hltp++pxuti*.

The first injectable formulation contains an effective amount of an enzyme-conjugated antibody or antibody fragment; The pharmaceutically acceptable injection vehicle preferably contains phosphorus at physiological pH and concentration. It is dissolved in acid buffered saline (PBS). The second injectable formulation contains less An effective amount of a soluble substrate conjugated to at least one diagnostic or therapeutic agent is (generally the same as that used for the first formulation) It is dissolved in a substance). This injectable preparation is specifically intended for human use. If it is, it is preferably sterile.

Reagents can also be used for antibody targeting to target sites using two suitable containers. It is advantageous if the kit is supplied as a therapeutic or diagnostic kit. The first container is Contains an effective amount of an antibody or antibody fragment covalently linked to an enzyme. second volume The device contains an effective amount of a soluble substrate conjugated to at least one therapeutic or diagnostic agent. include. Reagents may be lyophilized to extend storage stability or may be prepared in solution. It may be supplied in the form of a 100% polyester, containing customary preservatives, stabilizing agents, etc., if necessary.

Other optional components in such kits typically include buffers, labels, etc. a reagent, a radioisotope, a paramagnetic compound, a second antibody to aid clearance; There are regular syringes, columns, bottles, etc.

The methods of the invention are typically carried out by parenteral injection.

Various parenteral injections, e.g., intracorporeal (e.g., intraperitoneal), intravenous, dynamic, intrapleural, Intradural, intramuscular injection, intralymphatic, locally mobile, locally intralesional, subcutaneous, catheter perfusion, etc. However, it is not limited to these.

For cancer imaging and/or treatment, intravenous, dynamic, and pleural administration is typically used to , used for breast and white blood cell cancers. Intraperitoneal administration is advantageous for ovarian tumors. intradural Administration is advantageous for brain tumors and leukemia. Subcutaneous administration is recommended for Hodgkin's disease and lymphoma. , beneficial for lung cancer. Catheter perfusion is effective for metastatic cancer in the lungs and chest, and blastic cell carcinoma in the liver. It is valid. Intralesional administration is effective for lesions in the lungs and chest.

The above describes the antibody-enzyme conjugates and substrate-therapeutic or diagnostic agent conjugates of the present invention. This is an illustration of a general method of administration. Administration of two different conjugates may not be the same because the clearance pathways and biodistribution of cohabitants are generally different. It will be understood that this may not be necessary. For example, intraperitoneal administration of antibody-enzyme conjugates is advantageous for targeting ovarian tumors, but for imaging, radioisotope Preferably, the elementary-to-radical conjugate is administered intravenously. Because, in the latter case, the deposition rate This is because it is easy to control and it is easy to monitor the clearance speed. .

Antibody-enzyme conjugates are generally present in PBS, preferably especially for human use. is administered as an aqueous solution in sterile fluid. About 50 μg of antibody-enzyme conjugate It is convenient to administer a dosage unit of about 50 mg either in a single dose or in divided doses. be. However, lower or higher doses may be effective for specific cases. may be appropriate. The dose may be reduced and/or may require the use of antibodies from other animal species and/or hypoallergenic antibodies. It may become necessary.

i.e. to desensitize the patient, especially for therapeutic purposes, or, in particular, as a therapeutic prescription. or, in addition, where repeated administration is required for diagnostic procedures. . As a guideline when such careful treatment is required, human anti-mouse antibodies (HA MA) There is an increase in production. This can be quantified using immunoassay.

The IgG antibody localizes to the target site and is substantially removed from the mammal's circulatory system; It usually takes about 2 to 14 days, preferably about 2 to 14 days, before the drug-drug conjugate is ready for administration. It takes 5 to 14 days.

Corresponding localization of F(ab)2 and F(ab’)2 antibody fragments and clearance time is about 2 to 7 days, preferably 4 to 7 days, and For Fgb and Fxb' antibody fragments, about 1 to 3 days, preferably is 3 days. Other antibodies require a different time frame to localize to the target site. It might happen. The above time frame is also influenced by the presence of the conjugated enzyme It may happen. As an additional note here, if the antibody-enzyme conjugate is labeled, , thereby allowing localization and clearance to be monitored.

IgG is normally metabolized in the liver and to a lesser extent in the digestive system. F(ab)2o Normally, F(zb')2 is metabolized mainly in the kidneys, but also in the liver and the digestive system. Metabolized. Fib and Fxb' are mainly metabolized in the kidneys, but are metabolized in the liver. It is also metabolized in the digestive system.

Typically, prior to administration of the Motoga-enzyme conjugate, at least one of the antibody-enzyme conjugate doses is Approximately 0.0001% must be localized to the target site. The target of this zygote The higher the getting efficiency, the higher this percentage and the lower the dosage. It will be good if you give it.

Therefore, an effective amount of an antibody-enzyme conjugate means that the conjugate targets the antigen at the target site. target, thereby binding a sufficient amount of the enzyme. to convert a sufficient amount of soluble substrate-drug conjugate to product, resulting in , in an amount that results in accumulation of a diagnostically or therapeutically effective amount of the drug at the target site. Ru.

Substrate-therapeutic or diagnostic agent conjugates are generally administered as an aqueous solution in PBS. Ru. Again, if intended for human use, the solution should be sterile. Substrate-drug contact Coalescence causes the antibody-enzyme conjugate to localize to the target site and be substantially eliminated from the mammalian circulatory system. It is administered after sufficient time has elapsed for the drug to be removed.

The use of conjugates of boron-loaded carriers for thermal neutron activation therapy also Always do it in the same way. That is, non-targeting Moga-drug conjugates are removed. It is preferable to wait until neutron irradiation is performed before starting neutron irradiation. It goes like this For example, as can be seen from US Patent No. 4.624.846, can be accelerated by using a second antibody.

An effective amount of a substrate-drug conjugate is an amount sufficient to deliver an effective amount of the drug to the target site. It is also the case that the substrate is converted by the enzyme into a certain form of product, and the product is The amount of substrate is such that it accumulates at the target site. An effective amount of a therapeutic or diagnostic agent is The amount is sufficient for treatment and diagnosis of the target area.

If scintigraphic imaging is performed, the substrate-drug conjugate is bound to the substrate. radiolabeled species. This allows direct iodination even for chelates of radioactive metals. Alternatively, it may be a metalized compound. As a suitable gamma emitting isotope, , l-131, l-123, Tc-99m, In-+1j, G1-67 are listed. It will be done. Antibodies bind to antigens at target sites, and enzymes bind to substrate-drug conjugates. into a product, but the product is sufficiently small to allow imaging. It accumulates at the target site in large amounts. Once enough isotope is attached to the target site, If so, scan with a conventional planar gamma camera and /' or 5PECT gun. Either done by the Mac Camera or used externally or internally. The biological microlocation of tumors and infections is carried out by a manually operated gamma probe. , localize myocardial infarction, atherosclerotic plaque, or other target sites.

Scintigrams are usually created using a gamma imaging camera with one or more windows. Take a photo. This is used to detect energies in the 50-500 k+V range. . When using radioisotopes with high-energy beta or positron radiation, If so, an image camera with a suitable detector must be used. These are all This is common practice in this field.

As an example, polylysine oligomers are converted into succinimidyl p-isothiocyanate. Tobenzoate linker (reacts with multiple U.S. patented dextran oligomers) and then stabilize it with borohydride. Patients, e.g. cancer patients, are given anti-tumor A conjugate of a tumor antibody and a dextranase enzyme is injected, and the localization of the conjugate and the 7 days are allowed for clearance of the conjugate. Next, this substrate-key The rate agent conjugate was charged using filter-sterilized indium-111 ions in PBS. and inject it into the faithful. Accumulation of label is observed within approximately 3 hours, with background The clearance of the sign was almost completed in about 12-24 hours, so the image implementation.

Magnetic resonance imaging (MHI) is also performed in a similar manner to scintigraphic imaging, but The imaging agent is not a radioisotope and is an MRT enhancement enhancer. magnetic resonance phenomenon is , it is understood that it operates on a different principle from scintigraphy. Usually the generated signal is the relaxation of the magnetic moment of the proton in the hydrogen nucleus of the water molecule in the imaged region. Correlates with sum time. Magnetic resonance image enhancers increase the rate of relaxation, thereby improving the image The connection between water molecules in the area where the imaging agent accumulates and water molecules in other parts of the body Trust should be increased, the former leading to an increase in contrast and the latter leading to contrast. leading to a decrease in trust. Therefore, this phenomenon is concentration dependent and usually For certain paramagnetic materials, there is an optimum concentration for maximum efficiency. This optimum concentration The degree of sum-recovery, inversion-recovery methods, and various other strongly T1-dependent or T-dependent methods. 2 Depends on the imaging technique and the composition of the solvent in which the drug is dissolved or suspended. . These factors and their relative importance are known in the art. . For example, examples of compounds useful for MRI image enhancement include paramagnetic Gd(III) , Ea(lIll, Dr(1!I), Pr(III), Pg(IV), 1ln(II).

C+ (III), Co (fill, Fe (Ill), Cu (Ill), N1 (I ll, Ti(III) and V(IV) ions or groups (e.g. nitroki cids). These are paramagnetic ion chelators for ions, or , group addition factor j+! A group carrying a linker for dictl 1dde++ts) Combined with quality. Magnetic resonance image enhancers are commonly used to ensure patient safety and equipment safety. At magnetic field strengths that do not conflict with the design, the It must exist in quantity. The requirements for such drugs are within the field. It is well known that some substances act on water molecules in solvents, especially , P7kell (supra) and Range et al. (supra).

The same method used for scintigraphy is used for magnetic resonance imaging (MRI). is used. In the previous example, to achieve high contrast MRI enhancement, Since it is desirable to deliver a large number of paramagnetic ions to the target site, a large amount of chelating agent and relatively large amounts of antibody-enzyme conjugate and substrate-chelate conjugate. is administered, thereby attaching a high concentration of paramagnetic ions to the target site.

The treatment method of the present invention uses radioactive isotopes such as Y-90 or 1-IN (which can be used for both topical formulation and treatment, but the therapeutic efficacy (depending on the dose) or drugs such as adriamycin for cancer and gentamicin for infections. , or immunomodulators such as poly-IC, or from pokeweed. Biotoxins, such as mitogens, are bound to a substrate and an effective dose of the drug is delivered to the target site. This can be achieved by adhering. Treatments of the invention also include one or more of the boron-10 addition factor to the substrate, and once this boron-10 is attached to the target site, e.g. For example, if it adheres to a tumor, thermal neutron irradiation is applied to the tumor from the outside to remove the cancerous particles. This can also be achieved by destroying the cells. The boron-10 conjugate has It may also be labeled with a radioisotope chelate. In this way, sufficient boron addition factor localized to the target site and that most of the non-targeting boron-10 Before neutron irradiation, it can be verified that everything has left the circulatory system.

The dosage unit of the substrate-drug conjugate depends on many factors, each of which This is determined by a relatively simple method, such that the dose measurement (do+im+t+icl indicates the optimal value). It can be quantified in various ways. For initial dose measurements, the radiolabeled substrate - Using drug conjugates (if the drug itself is not a radioisotope) to The amount of drug deposited at the site, the deposition rate, the clearance rate, and non-targeting contact. It is convenient to quantify the biodistribution of coalescence. Estimating the amount of enzyme localized at the target site The use of labeled antibody-enzyme conjugates to determine dose measurements is also effective. It is.

Dose-finding studies are generally performed first in animal models and then, if possible, It is necessary to conduct a series of clinical trials. This determines the optimal dose of the substrate-drug conjugate, Proximity of site (*ce++xibilit7), administration method, enzyme turnover rate, Desired dose of drug to site, non-targeted This is to know as a function of the clearance rate of the conjugate. This relationship is difficult to predict. Combs and methods for optimization are also within the routine skill of the clinician.

Without going into further detail, a person skilled in the art can use the foregoing description to understand , it is considered possible to take full advantage of the present invention. Therefore, below The individual preferred embodiments are presented merely by way of illustration and are not intended to be interpreted as meaning any meaning. However, it does not limit the other parts of the present disclosure.

In the examples below, all temperatures are expressed in degrees Celsius, uncorrected; All parts and percentages are by weight, unless otherwise indicated.

Example 1 Preparation of methotrexate/aminodextran conjugate (a) of methotrexate activation In a dry reaction bottle, add 45.411 g of methotrex dissolved in 1 ml of anhydrous DMF. Sate (0.1 mmol, 51 gm5) was added with a syringe.

N-hydroxysuccinimide (23 mg, 0.2 mmol, 51 gml) and 1 dissolved in 750 μ+ anhydrous DMF. , 3-dicyclohexylcarbodiimide (41.5 mg, 0.2 tact, Sigma) was further added. The reaction mixture was incubated in the dark at room temperature for 16 hours. Stirred under anhydrous conditions. Centrifuge the white precipitate, put the clear solution into a sealed stopper, and store at -20°C. Saved with.

(b) Reaction with aminodextran Aminodextran (lomg, 2.5! lO’ not) was added to 2 ml of P Dissolved in BS, pH 7.2. Activated MT X (125tLO’ mmol) was added gradually. The solution was stirred at room temperature for 5 hours and then washed with Sephadex G-25 color. It was purified using Collect void volume and further dialyze against reaction buffer did. After lyophilization, 2.1 mg of product was obtained (21% yield). Methotrexe The uptake of 38 methotrexate/dextrose was determined by absorption at 370 nm. It was determined that Tran.

Example 2 Preparation of chelating agent-polylysine/dextran conjugate Polylysine (MW 15.0 00) in an amount sufficient to bind an average of 5 DTPAs to this polylysine. Succinimidyl p-isothiocyanate benzoate of minomethyl-DTPA Derivative (succinimidyl benzoate forms DTPA through thiourea linkage) (combined with). The resulting product was added at a concentration of approx. Pre-acidify lightly with periodate just enough to generate two aldehyde groups. dextran oligomer (MW 1.500). This reaction , sufficient to load approximately 3-5 dextran units onto the phorylysin-DTPA. Do it in the right amount. Furthermore, it is stabilized with boron hydride and 5chill! Base binding and survival Reduces aldehyde groups.

Ebilubicin is given to horses intravenously over a period of several weeks. Collect urine and ion exchange urine Ebilubicin glucuronide was isolated by chromatography, and then Purify by ram chromatography and/or HPLC.

(A) Practical monoconing enzyme-antibody preparation The product is prepared as follows. That is, the carbohydrate moiety of anti-CEArgG was gently oxidized with periodate, and then this oxidized 1 gG was oxidized with dextranase ( From Penieillium genus, Wo+thiBton Biochemicx Antibody-enzyme An elementary conjugate is produced which is then stabilized with borohydride as usual.

This conjugate can be radiolabeled with l-131 by conventional methods.

(B) In the same manner as in A above, anti-leukocyte TgG was treated with glucuronidase (derived from bovine liver, Combines with Wo+lhingon).

Anti-CEA IgG/Dex dissolved in PBS for a human with small cell carcinoma of the right lung A sterile, pyrogen-free solution containing 5 mg of the tranase conjugate was injected intravenously. child A solution of l-131 was prepared according to this Example 4(A) and labeled with l-131. There is.

After 5 days, the conjugate was well localized to the lungs and largely cleared from the patient's circulatory system. child This was confirmed by daily scintigraphic scanning.

Sterile, pyrogen-free PBS solution of MTX/aminodextran (Example 1) (containing the conjugate somg) was injected intravenously every day for the next 4 days. So Afterwards, radioimmunodetection using l-123-anti-CEAFab showed a significant tumor reduction. I found out that there are a few.

Example 6 Anti-lymphoma 1gG-glucuronidase conjugate 5mg in human patients with lymphoma A sterile, pyrogen-free PBS solution containing PBS was injected intravenously. This zygote is carried out Prepared according to Example 4(b) and labeled with 1-131. For gamma scanning After 6 days, this conjugate is well localized at the target site and circulating It was almost completely removed from the system.

Next, the patient is given a sterile, pyrogenic solution containing lomg of evirubicin glucuronide. A non-containing PBS solution was injected intravenously. This solution was prepared according to Example 3. and intravenous injections were given daily for the next 4 days. Then, radioimmunodetection It was found that lymphoma was significantly reduced.

Human patients with rectal cancer containing 5 mg of anti-CEA-IgG/dextranase conjugate , sterile, pyrogen-free PBS solution was injected intravenously. This zygote was produced in Example 4 (A ) was prepared according to

Seven days later, patients received lff-111 labeled polydine-DTPA/dextran conjugate. 5 Sterile, pyrogen-free PBS containing mci The solution was injected intravenously. This bonded body was prepared according to Example 2, and /=In-1ll was prepared according to Example 2. Loaded. After 24 hours, scintigraphic images show that 100 anti-CEA-IgG was used in human patients with ascending colon tumors who had accumulation of radioisotopes in large amounts. /dextranase conjugate in sterile, pyrogen-free PBS solution. I gave it an intravenous injection. This conjugate was prepared according to Example 4(A). After 7 days, the patient , containing 50 hg of Gd(III)-loaded polylysine-DTPA/dexran conjugate , sterile, pyrogen-free PBS solution was injected intravenously. This complex was prepared according to Example 2. It was prepared as follows.

Two more days later, an MRI image was taken that showed the tumor, which was surrounded by surrounding tissue. It was well differentiated from the texture.

Reactants of the invention, generally and specifically described, used in the examples above and/or may be repeated with permutations of operating conditions with similar results. came.

From the above description, a person skilled in the art would be able to confirm the essential features of the present invention. Various changes and modifications may be made to the present invention without departing from its spirit or scope. It is possible to add positives and change it to suit various usages and conditions.

Copy and translation of written amendment) Submission (Article 184-8 of the Patent Law) June 11, 1992 1. Indication of patent application: PCT/US 89105441, title of invention: Diagnostic agent or or antibody targeting of therapeutic drugs 3, patent applicant Address: Warren, New Jersey 07060, United States E.N. 4918, Mount-Bethel Road, 100 Name Immunomede Six Incorporated 4, Manager 1-1 Shinjuku, Shinjuku-ku, Tokyo No. 14 Yamada Building 5, Date of submission of written amendment: October 3, 1991 Scope of claims 1′, a method for targeting a diagnostic or therapeutic agent to a target site, the method comprising: (a) to localize to a target site and to provide enzymatic activity to said site; parenterally injecting into the mammal an amount of the antibody-enzyme conjugate effective to the antibody is reactive with at least one antigen present at the target site, and ( b) the antibody-enzyme conjugate is localized to the target site and is located in the circulatory system of the mammal; after sufficient time has elapsed to be substantially removed from the site. parenterally injecting a sufficient amount of the soluble drug-drug conjugate into said mammal, with the proviso that: and the conjugate is converted by the enzyme into at least one diagnostic or therapeutic agent. A product containing the therapeutic agent can be formed, said product accumulating at said target site. In addition, the drug-drug conjugate enables effective treatment or diagnosis. comprising a substrate for said enzyme, both conjugated to one said diagnostic or therapeutic agent; , said enzyme and any of the enzymes having similar activity with respect to said substrate-enzyme conjugate. Either route of administration or in vivo administration of the substrate-drug conjugate in the mammal. an amount that prevents targeting and accumulation of the drug at non-target sites along the distribution route; So it doesn't exist, A method comprising the steps described above.

2. The antibody in the antibody-enzyme conjugate may be used to treat tumors, infections or parasitic lesions, Phosphate clots, myocardial infarction, atherosclerotic plaques, non-cancerous cell damage-related sites, or produced by or associated with damage-related areas of injured normal cells 2. The method of claim 1, wherein the method specifically binds to an antigen that binds to an antigen.

3. The enzyme in the antibody-enzyme conjugate is a protease, a glycosidase, The method according to claim 1, which is glucuronidase or esterase.

4. The enzyme is dextranase, cellulase, or glucuronidase The method according to claim 3.

5. The method according to claim 1, wherein the drug is a diagnostic agent.

6. The drug emits gamma radiation in the energy range of 5 (1-500 Ku) 6. The method according to claim 5, wherein the radioactive isotope is a radioactive isotope.

7. The drug according to claim 5, wherein the drug is a paramagnetic ion for magnetic resonance image enhancement. Method.

8. The method of claim 1, wherein the agent is a therapeutic agent.

9. The drug is a beta- or alpha-emitting radioisotope, a drug , toxins, boronates, vasodilators, cytokines, photosensitizers, or radiation 9. The method according to claim 8, which is a radiation sensitizer.

10. The parenteral injection may be intrabody cavity, intravenous, intraarterial, intraperitoneal, intradural, or lymphatic. Performed by intraluminal, intramuscular, intralesional, subcutaneous, or catheter perfusion routes , the method of claim 1.

11. The method according to claim 1, wherein the substrate is a low molecular weight compound.

12. The method according to claim 11, wherein the substrate is a glucuronide conjugate of the drug. Method.

13. The method of claim 1, wherein the substrate is a polymer.

14. The substrate is dextran, aminodextran, carboxymethyl 14. The method according to claim 13, wherein the method is lulose or a polypeptide.

15. The enzyme is a dextranase or a cellulase, and the enzyme is a dextranase or a cellulase; the substrate-drug conjugate comprises a non-adsorbable aminodextran or polylysine carrier; , to which at least one drug molecule or ion is bound; Further, the carrier contains at least one soluble dextra which is a substrate for the enzyme. claim 13, wherein the methyl cellulose oligomer is attached to The method described in.

16. The substrate-drug conjugate contains a non-chemical polymer, and this polymer contains a small amount of At least one substrate oligomer is attached to the oligomer, at least one 14. The method of claim 13, wherein the drug molecules or ions are bound.

17. Clearance of the antibody-enzyme conjugate from the circulatory system or at the target site To monitor the localization of the conjugate, the antibody-enzyme conjugate is further attached to or for attachment to a sexual isotope or magnetic resonance image enhancer The method according to claim 1, wherein the method is modified to.

18. Clearance of the substrate-drug conjugate from the circulatory system or at the target site The substrate-drug conjugate is further irradiated to monitor the accumulation of the conjugate. conjugated to a sexual isotope, magnetic resonance image enhancer, or other label; or modified for binding.

19. The method according to claim 1, wherein the mammal is a human.

20. For use in humans to target therapeutic or diagnostic agents to target sites. a dual sterile injectable preparation for use, comprising: (a) a pharmaceutically acceptable sterile injectable formulation; in a vehicle to localize to and provide enzymatic activity to the target site. a first sterile injectable solution containing an amount of the antibody-enzyme conjugate effective to the antibody is reactive with at least one antigen at the target site, and (b) an agent containing an effective amount of a soluble substrate-drug conjugate to attach to said site; 2 sterile injectable solution, provided that the conjugate has been converted by the enzyme to at least 1 It is possible to form a product containing two diagnostic or therapeutic agents, said substrate-drug The agent conjugate is a substrate of the enzyme linked to at least one diagnostic or therapeutic agent. having similar activity with respect to said enzyme and said substrate-enzyme conjugate. Any of these enzymes may be used in mammals to administer the substrate-drug conjugate or targets and accumulates the drug at non-target sites along the biodistribution pathway. The substrate-drug conjugate is not present in an interfering amount, and the substrate-drug conjugate is a pharmaceutically acceptable sterile injectable The first and second injection solutions are dissolved in a vehicle for injection. formulation.

21. Human diagnostics for targeting therapeutic or diagnostic agents to target sites or a kit for use in treatment, (a) to localize to a target site and to provide enzymatic activity to said site; a first sterile container containing an amount of an antibody-enzyme conjugate effective to is reactive with at least one antigen present at the target site; and (b) a second container containing an effective amount of soluble substrate-drug conjugate to attach to said site; a bacterial container, provided that said conjugate is transformed by said enzyme to produce at least one diagnostic a drug or a therapeutic agent, the substrate-drug conjugate comprises a substrate for said enzyme conjugated to at least one diagnostic or therapeutic agent; wherein an enzyme having similar activity with respect to said enzyme and said substrate-drug conjugate; Either route of administration or in vivo administration of the substrate-drug conjugate in mammals. Preventing targeting and accumulation of the drug at non-target sites along the internal distribution pathway It is not inherent in quantity, A kit comprising the first and second containers.

22. The therapeutic or diagnostic agent contains a small amount (at least one boron-added factor, drug, Toxins, radioisotopes, nuclear magnetic resonance image enhancers, vasodilators, cytokines, The kit according to claim 21, which is a radiation sensitizer or a photosensitizer.

23. The antibody-enzyme conjugate is further subjected to radioisotope or magnetic resonance imaging. according to claim 21, which is conjugated to or modified for conjugation to an enhancer. Kit as described.

24. The substrate-drug conjugate further contains a radioisotope, a magnetic resonance image enhancer, , or conjugated to or modified for conjugation to other labels. 22. The kit of claim 21.

25. The antibody-enzyme conjugate provided in step (a) is present at the target site. a first binding site specific for the antigen; and a first binding site for the enzyme that does not interfere with enzymatic activity. a bispecific antibody with a second binding site specific for the above epitope or comprises an antibody fragment, said bispecific antibody or antibody fragment comprising: non-covalently bound to the enzyme at the second binding site to bind the antibody-enzyme conjugate; A method according to claim 1, characterized in that:

26. For use in humans to target therapeutic or diagnostic agents to target sites. a sterile injectable preparation for use in a pharmaceutically acceptable sterile injectable vehicle, the preparation comprising: (a) a pharmaceutically acceptable sterile injectable vehicle; of claim 25, in an amount effective for targeting and enzymatic activity, dissolved in a (b) a first sterile injectable solution containing an antibody-enzyme conjugate as described above; a second sterile injectable solution containing an effective amount of a soluble substrate-drug conjugate to adhere; provided that the conjugate is converted by the enzyme into at least one diagnostic agent or It is possible to form a product containing a therapeutic agent, wherein the substrate-drug conjugate comprising a substrate for said enzyme conjugated to at least one diagnostic or therapeutic agent, wherein: any of the enzymes and enzymes with similar activity with respect to the substrate-enzyme conjugate; However, in humans, the route of administration or biodistribution of the substrate-drug conjugate is endogenous non-target sites along the route in amounts that would interfere with targeting and accumulation of the drug. First, the substrate-drug conjugate is dissolved in a pharmaceutically acceptable sterile injectable vehicle. A formulation comprising the first and second injection solutions.

27. Human diagnostics for targeting therapeutic or diagnostic agents to target sites A kit for use in cutting or treating (a) an amount effective for targeting and enzymatic activity of the antibody-enzyme of claim 25; a first sterile container containing the elementary conjugate; and (b) an agent containing an effective amount of a soluble substrate-drug conjugate to attach to said site; 2 sterile containers, provided that said conjugate has been converted by said enzyme to contain at least one A product containing a diagnostic or therapeutic agent can be formed, wherein the substrate-agent The conjugate contains a substrate for said enzyme conjugated to at least one diagnostic or therapeutic agent. comprising, wherein said enzyme and said substrate-drug conjugate have similar activities. Any of these enzymes may be used in humans by the route of administration or bioavailability of the substrate-drug conjugate. Preventing targeting and accumulation of the drug at non-target sites along the biodistribution pathway a kit characterized in that the kit comprises the first and second containers in an amount that does not contain the first and second containers; .

28. The therapeutic or diagnostic agent comprises at least one boron addition factor, drug, Toxins, radioisotopes, nuclear magnetic resonance image enhancers, vasodilators, cytokines, The kit according to claim 27, which is a radiation sensitizer or a photosensitizer.

29. A method for targeting a diagnostic or therapeutic agent to a target site, comprising: (a) A first binding site specific to the antigen present in the target site and a first binding site specific to the enzyme providing a bispecific antibody or antibody fragment with a second binding site (b) an effective amount of said antibody or antibody fragment; (c) injecting said antibody or antibody fragment parenterally into a mammal; sufficient to localize to the target site and be substantially cleared from the mammal's circulatory system. After a period of time, the localized antibody binds to the enzyme and the antibody-enzyme contact occurs. An enzymatically effective amount of said enzyme is administered non-enzymatically to said mammal such that the conjugate forms in situ. injecting orally; (d) further comprising a soluble amount effective to adhere to said site; parenterally injecting a sex substrate-drug conjugate into said mammal, with the proviso that said conjugate The body is converted by said enzyme into a product containing at least one diagnostic or therapeutic agent. The product can accumulate at the target site to provide effective therapy. or diagnosis, and the substrate-drug conjugate is comprising a substrate for said enzyme conjugated to said diagnostic or therapeutic agent; and enzymes with similar activity with respect to said substrate-enzyme conjugate. In mammals, along the administration route or biodistribution route of the drug-drug conjugate. The drug is not present at non-target sites in amounts that prevent dasetting and accumulation of the drug. A method comprising the steps described above.

30. The antibody or antibody fragment in the antibody-enzyme conjugate may be used to treat tumors, infections, etc. or parasitic lesions, fibrin clots, myocardial infarction, atherosclerotic plaques, non-cancerous cells. , or produced by damaged normal cells, or damaged associated parts of damaged normal cells, or 30. The method of claim 29, wherein the method specifically binds to a relevant antigen.

31. The enzyme in the antibody-enzyme conjugate is a protease, a glycosidase, or a glycogenase. The method according to claim 29, which is lucuronidase or esterase.

32. The therapeutic or diagnostic agent contains at least one boron addition factor, a drug, Toxins, radioisotopes, nuclear magnetic resonance image enhancers, vasodilators, cytokines, 30. The method according to claim 29, which is a radiation sensitizer or a photosensitizer.

33. The enzyme is a dextranase or a cellulase, and the enzyme is a dextranase or a cellulase; the substrate-drug conjugate comprises a non-adsorbable aminodextran or polylysine carrier; , to which at least one drug molecule or ion is bound; Furthermore, the carrier contains at least one soluble dextrin, which is a substrate for the enzyme. 29 . 29 . 29 . The method described in.

34. The method of claim 29, wherein the mammal is a human.

35. For use in humans to dase a therapeutic or diagnostic agent to a target site. A sterile injectable preparation for use comprising: (a) a drug specific for an antigen present at a target site; a dase protein having a first binding site and a second binding site specific for the enzyme; a first sterilization containing an amount of bispecific antibody or antibody fragment effective for Injectable solution, provided that the antibody or antibody fragment is in a pharmaceutically acceptable sterile injection solution. dissolved in the injection vehicle; (b) a second enzyme containing an amount of said enzyme effective for enzymatic activity at said target site; bacterial injection, provided that the enzyme is dissolved in a pharmaceutically acceptable sterile injection vehicle. and (C) an effective amount of a soluble drug agent to attach to said site. a third sterile injectable solution containing a conjugate, provided that said conjugate is converted by said enzyme; to form a product containing at least one diagnostic or therapeutic agent; The agent conjugate comprises the enzyme conjugated to at least one of the diagnostic or therapeutic agents. comprising a substrate, said substrate-enzyme conjugate being in a pharmaceutically acceptable sterile injectable vehicle. is dissolved in wherein said enzyme and an enzyme having similar activity with respect to said substrate-enzyme conjugate; Either route of administration or in-vivo distribution of the drug-drug conjugate in humans. at non-target sites along the fabric path in an amount that prevents derzetting and accumulation of the drug. and the substrate-drug conjugate is in a pharmaceutically acceptable sterile injectable vehicle. characterized by comprising the first, second and third injection solutions dissolved in a vehicle. Preparations that contain

36. Human diagnostics for administering therapeutic or diagnostic agents to target sites A kit for use in cutting or treating (a) A first binding site specific to the antigen present in the target site and a first binding site specific to the enzyme an effective amount of a bispecific antibody or a first sterile container containing the antibody fragment; (b) a second enzyme containing an amount of said enzyme effective for enzymatic activity at said target site; bacteria container, and (C) an agent containing an effective amount of a soluble substrate-drug conjugate to attach to said site; 3 of the sterile container, provided that said conjugate has been converted by said enzyme to at least one A product containing a diagnostic or therapeutic agent can be formed, wherein the substrate-agent The conjugate contains a substrate for said enzyme conjugated to at least one diagnostic or therapeutic agent. comprising, wherein said enzyme and said substrate-drug conjugate have similar activities. Any of these enzymes may be used in humans by the route of administration or bioavailability of the substrate-drug conjugate. Preventing targeting and accumulation of the drug at non-target sites along the biodistribution pathway said first, second, and third containers are not present in an amount that is kit.

37. The therapeutic or diagnostic agent contains at least one boron addition factor, a drug, Toxins, radioisotopes, nuclear magnetic resonance image enhancers, vasodilators, cytokines, 37. The kit according to claim 36, which is a radiation sensitizer or a photosensitizer.

international search report

Claims (1)

[Claims] 1. A method of targeting a diagnostic or therapeutic agent to a target site, the method comprising: (a) parenterally injecting into a mammal an amount of an antibody-enzyme conjugate effective for targeting and enzymatic activity, provided that said antibody is reactive with at least one antigen present at the target site, and (b) said antibody-enzyme conjugate is localized at the target site and substantially cleared from the circulatory system of the mammal. parenterally injecting into said mammal a sufficient amount of a soluble substrate-drug conjugate to adhere to said site after a sufficient period of time has elapsed for said conjugate to be converted by said enzyme; to form a product comprising at least one diagnostic or therapeutic agent, said product accumulating at said target site to enable effective treatment or diagnosis, and said substrate-drug conjugate body is small comprising a substrate for said enzyme conjugated to at least one said diagnostic or therapeutic agent, wherein said enzyme and enzymes having similar activity with respect to said substrate-enzyme conjugate; Either route of administration or biological route of administration of the substrate-drug conjugate in the mammal. A method comprising the step of: not being present at a non-target site along the internal distribution pathway in an amount that would interfere with targeting and accumulation of the agent. 2. The antibody in the antibody-enzyme conjugate is directed against tumors, infected or parasitic lesions, fibrillar A protein that specifically binds to antigens produced by or associated with blood clots, myocardial infarction, atherosclerotic plaques, non-cancerous cells, or injured normal cells. The method described in claim 1. 3. The enzyme in the antibody-enzyme conjugate may be a protease, a glycosidase, or a glucosidase. The method according to claim 1, wherein the method is a lonidase or an esterase. 4. The method according to claim 3, wherein the enzyme is dextranase, cellulase, or glucuronidase. 5. 2. The method of claim 1, wherein the agent is a diagnostic agent. 6. 6. The method of claim 5, wherein the agent is a gamma-emitting radioisotope that emits in the 50-500 KeV energy range. 7. 6. The method of claim 5, wherein the agent is a paramagnetic ion for magnetic resonance image enhancement. 8. 2. The method of claim 1, wherein the agent is a therapeutic agent. 9. 9. The method of claim 8, wherein the agent is a beta- or alpha-emitting radioisotope, a drug, a toxin, a boronated factor, a vasodilator, a cytokine, a photosensitizer, or a radiosensitizer. 10. The parenteral injection may be intracorporeal, intravenous, intraarterial, intraperitoneal, intradural, or lymphatic. 2. The method of claim 1, wherein the method is performed by intravenous, intramuscular, intralesional, subcutaneous, or catheter perfusion routes. 11. 2. The method of claim 1, wherein the substrate is a low molecular weight compound. 12. 12. The method of claim 11, wherein the substrate is a glucuronide conjugate of the drug. Law. 13. 2. The method of claim 1, wherein the substrate is a polymer. 14. The substrate is dextran, aminodextran, carboxymethyl cell 14. The method according to claim 13, wherein the method is a loose or a polypeptide. 15. the enzyme is a dextranase or a cellulase, wherein the enzyme is a dextranase or a cellulase; The drug-drug conjugate comprises a non-substrate aminodextran or polylysine carrier to which at least one said drug molecule or ion is bound; 14. The method of claim 13, wherein the carrier is further bound to at least one soluble dextran or carboxymethyl cellulose oligomer that is a substrate for the enzyme. 16. The substrate-drug conjugate includes a non-substrate polymer, the polymer having a small amount of 14. The method of claim 13, wherein at least one substrate oligomer is attached to which at least one drug molecule or ion is attached. 17. Clearance of the antibody-enzyme conjugate from the circulation or its target site 2. The antibody-enzyme conjugate is further conjugated to or modified for conjugation to a radioisotope or a magnetic resonance image enhancer to monitor the localization of the conjugate. Method described. 18. Clearance of the substrate-drug conjugate from the circulatory system or its target site The substrate-drug conjugate may be further conjugated to a radioisotope, magnetic resonance image enhancer, or other label to monitor the accumulation of the conjugate. 2. The method of claim 1, wherein the method is modified for conjugation. 19. 2. The method of claim 1, wherein the mammal is a human. 20. A dual sterile injectable formulation for human use for targeting a therapeutic or diagnostic agent to a target site, comprising: (a) targeting and enzymatic activity in a sterile pharmaceutically acceptable injectable vehicle; containing an effective amount of the antibody-enzyme conjugate. a first sterile injectable solution comprising: wherein said antibody is reactive with at least one antigen of the target site; and (b) an effective amount of a soluble substrate-drug conjugate to attach to said site. a second sterile injectable solution containing, with the proviso that said conjugate is not mediated by said enzyme; wherein the substrate-drug conjugate comprises the oxygen substrate bound to at least one diagnostic or therapeutic agent. , wherein either the enzyme and the enzyme with similar activity with respect to the substrate-oxygen conjugate are present at non-target sites along the route of administration or biodistribution of the substrate-drug conjugate in the mammal. The drug type The substrate-drug conjugate is not present in amounts that would interfere with targeting and accumulation of the drug. said first and second injections being dissolved in a pharmaceutically acceptable sterile injectable vehicle; A preparation characterized by containing an injection liquid. 21. A kit for use in human diagnosis or therapy for targeting a therapeutic or diagnostic agent to a target site, the kit comprising: (a) an amount of an antibody-enzyme conjugate effective for targeting and enzymatic activity; 1, provided that said antibody is reactive with at least one antigen present at its target site, and (b) contains an effective amount of a soluble substrate-drug conjugate to attach to said site. a second sterile container containing said conjugate, wherein said conjugate has been converted by said enzyme to at least one wherein the substrate-drug conjugate comprises the oxygen substrate conjugated to at least one diagnostic or therapeutic agent, wherein the enzyme and having similar activity with respect to said substrate-drug conjugate. Any of these enzymes may be used in mammals to administer the substrate-drug conjugate or a drug comprising said first and second containers that do not reside in an amount that would interfere with targeting and accumulation of said agent at a non-target site along a biodistribution pathway. t. 22. The therapeutic or diagnostic agent may include at least one boron-added factor, drug, or poison. element, radioactive isotope, nuclear magnetic resonance image enhancer, vasodilator, cytokine, radiation The kit according to claim 21, which is a radiation sensitizer or a photosensitizer. 23. The antibody-enzyme conjugate is further treated with a radioisotope or a magnetic resonance image enhancer. 22. The compound according to claim 21, which is conjugated to or modified for conjugation to a strong agent. Kit included. 24. 22. The kit of claim 21, wherein the substrate-drug conjugate is further conjugated or modified for conjugation to a radioisotope, magnetic resonance image enhancer, or other label. 25. Before the antibody-enzyme conjugate provided in step (a) is present at the target site. a bispecific antibody or antibody fragment having a first binding site specific for said antigen and a second binding site specific for an epitope on said enzyme that does not interfere with enzyme activity. , the bispecific antibody or antibody fragment is non-covalently linked to the enzyme at a second binding site to form the antibody-enzyme conjugate. The method according to claim 1, comprising: 26. A sterile injectable formulation for human use for targeting a therapeutic or diagnostic agent to a target site, the formulation comprising: (a) a pharmaceutically acceptable sterile injectable vehicle; a first sterile injectable solution containing an effective amount of the antibody-enzyme conjugate of claim 25 for targeting and enzymatic activity, dissolved in a a second sterile injectable solution containing an effective amount of the soluble substrate-drug conjugate to and the conjugate is converted by the enzyme into at least one diagnostic or therapeutic agent. The substrate-drug conjugate comprises at least both comprising a substrate for said enzyme conjugated to one diagnostic or therapeutic agent, wherein said and oxygen having similar activity with respect to the substrate-enzyme conjugate in humans along the route of administration or biodistribution of the substrate-drug conjugate. said first and second substances are not present at a non-target site in an amount that would interfere with targeting and accumulation of said drug, and said substrate-drug conjugate is dissolved in a pharmaceutically acceptable sterile injectable vehicle. A preparation comprising an injection solution of. 27. 26. A kit for use in human diagnosis or therapy for targeting a therapeutic or diagnostic agent to a target site, comprising: (a) an effective amount of the antibody-enzyme of claim 25 for targeting and enzymatic activity; a first sterile container containing an elementary conjugate; and (b) a second sterile container containing an effective amount of a soluble substrate-drug conjugate to adhere to said site, provided that said conjugate is at least one converted by an enzyme wherein the substrate-drug conjugate comprises a substrate for the enzyme conjugated to at least one diagnostic or therapeutic agent; and having similar activity with respect to said substrate-drug conjugate. Any of these enzymes prevents the targeting and accumulation of the drug at non-target sites along the route of administration or biodistribution of the substrate-drug conjugate in humans. A kit comprising said first and second containers, wherein said first and second containers do not contain said first and second containers in said quantities. 28. The therapeutic or diagnostic agent may include at least one boron-added factor, drug, or poison. element, radioactive isotope, nuclear magnetic resonance image enhancer, vasodilator, cytokine, radiation The kit according to claim 27, which is a radiation sensitizer or a photosensitizer. 29. A method for targeting a diagnostic or therapeutic agent to a target site, the method comprising: (a) a first binding site specific for an antigen present in the target site and a second binding site specific for an enzyme; (b) parenterally injecting a targeting effective amount of said antibody or antibody fragment into a mammal; (c) said antibody or antibody fragment has sufficient to localize to the target site and be substantially cleared from the mammal's circulatory system. After a period of time, the localized antibody binds to the oxygen and the antibody-enzyme contact. (d) parenterally injecting into said mammal an enzymatically effective amount of said enzyme so as to form a conjugate in situ; parenterally injecting a sexual substrate-drug conjugate into said mammal, with the proviso that said conjugate is converted by said enzyme into a product containing at least one diagnostic or therapeutic agent. the substrate-drug conjugate is capable of forming a product that accumulates at the target site to enable effective treatment or diagnosis, and the substrate-drug conjugate is comprising a substrate for said enzyme conjugated to said diagnostic or therapeutic agent, wherein said oxygen and and oxygen having similar activity with respect to the substrate-oxygen conjugate along the route of administration or biodistribution of the substrate-drug conjugate in the mammal. is not present at non-target sites in amounts that would interfere with targeting and accumulation of the drug. A method comprising the steps described above. 32. The antibody or antibody fragment in the antibody-enzyme conjugate may also be used to treat tumors, infections, etc. or a parasitic lesion, a fibrin clot, a myocardial infarction, an atherosclerotic plaque, a non-cancerous cell, or an antigen produced by or associated with an injured normal cell. Method. 33. The enzyme in the antibody-enzyme conjugate may be a protease, a glycosidase, a glucose 32. The method according to claim 31, which is clonidase or esterase. 34. The therapeutic or diagnostic agent comprises at least one boron-added factor, drug, or poison. element, radioactive isotope, nuclear magnetic resonance image enhancer, vasodilator, cytokine, radiation 32. The method according to claim 31, which is a radiation sensitizer or a photosensitizer. 35. wherein said oxygen is dextranase or cellulase; The drug-drug conjugate comprises a non-substrate aminodextran or polylysine carrier to which at least one said drug molecule or ion is bound; 32. The method of claim 31, wherein the carrier is further conjugated to at least one soluble dextran or carboxymethyl cellulose oligomer that is a substrate for the enzyme. 36. 32. The method of claim 31, wherein the mammal is a human. 37. A sterile injectable formulation for human use for targeting a therapeutic or diagnostic agent to a target site, comprising: (a) a first binding site specific for an antigen present at the target site; a targeting protein with a specific second binding site; a first sterile infusion containing an amount of bispecific antibody or antibody fragment effective for (b) a second injectable solution containing an amount of said oxygen effective for enzymatic activity at said target site; Extinction bacterial injection, provided that the enzyme is dissolved in a pharmaceutically acceptable sterile injection vehicle. and (c) a third sterile injectable solution containing an effective amount of a soluble substrate-drug conjugate to attach to said site, provided that said conjugate has been converted by said enzyme to at least one forming a product containing two diagnostic or therapeutic agents, said substrate-drug wherein the agent conjugate comprises a substrate for said enzyme coupled to at least one said diagnostic or therapeutic agent, said substrate-enzyme conjugate being dissolved in a pharmaceutically acceptable sterile injectable vehicle. In humans, both the enzyme and the enzyme having similar activity with respect to the substrate-enzyme conjugate are administered by the route of administration or in vivo of the substrate-drug conjugate. at non-target sites along the fabric path in an amount that prevents targeting and accumulation of the drug. and the substrate-drug conjugate is in a pharmaceutically acceptable sterile injectable vehicle. A preparation comprising the first, second and third injection solutions dissolved in a vehicle. 38. A kit for use in human diagnosis or treatment for targeting a therapeutic or diagnostic agent to a target site, the kit comprising: (a) a first binding site specific for an antigen present in the target site; and an enzyme. a targeting effective amount of a bispecific antibody or (b) a second sterile container containing an amount of said enzyme effective for enzymatic activity at said target site; (c) a third sterile container containing an effective amount of a soluble substrate-drug conjugate to attach to said site, provided that said conjugate has been converted by said enzyme to at least one wherein the substrate-drug conjugate comprises a substrate for the enzyme conjugated to at least one diagnostic or therapeutic agent; and having similar activity with respect to said substrate-drug conjugate. Any of these enzymes prevents the targeting and accumulation of the drug at non-target sites along the route of administration or biodistribution of the substrate-drug conjugate in humans. A kit comprising said first, second, and third containers, wherein said first, second, and third containers do not contain the same amount. 39. The therapeutic or diagnostic agent may include at least one boron-added factor, drug, or poison. element, radioactive isotope, nuclear magnetic resonance image enhancer, vasodilator, cytokine, radiation The kit according to claim 38, which is a radiation sensitizer or a photosensitizer.